Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.


Introduction
Type III secretion systems (T3SS) are found in many Gram-negative pathogens and symbionts of animals and plants, including members of the genera Salmonella, Shigella, Yersinia, Rhizobium, Escherichia, and Pseudomonas [1]. These systems, evolutionarily related to bacterial flagella, are similar to molecular needles that span the inner membrane, periplasmic space, outer membrane, and host cell membrane to translocate proteins, called effectors, into the cytosol of the host cell cytoplasm [2]. Bacteria of the species Salmonella enterica infect humans and other animals causing a variety of diseases, including gastroenteritis, abortion, and typhoid fever, depending on the serotype of S. enterica and the host involved. These bacteria possess two distinct T3SSs, encoded in Salmonella Pathogenicity Islands (SPIs), which secrete about 40 effectors [3,4]. SPI1-encoded T3SS (T3SS1) is important for the invasion of host cells [5]. Once inside the cell, Salmonella survives and replicates in a modified phagosome known as the Salmonella-containing vacuole (SCV) [6]. The SPI2encoded T3SS (T3SS2) is expressed in response to environmental cues that are found in the SCV. Effectors secreted by this system are essential to maintain SCV and to allow Salmonella survival within phagocytic cells [7]. These T3SS have been mainly studied in S. enterica serovar Typhimurium. This serovar, which causes gastroenteritis in humans, produces a systemic disease in susceptible mice that is similar to typhoid fever caused in humans

Glucosylceramidase Activity of SrfJ
The similarity between SrfJ and human glucosylceramidase led us to explore the enzymatic activity of this Salmonella effector. For this purpose, the 4-methylumbelliferyl-β-D-glucopyranoside substrate was used. Hydrolysis of this substrate results in a product with emission at the blue region of the fluorescence spectrum. The purified 6His-SrfJ in this assay showed a glucosidase activity of 0.83 U/mg, significantly higher than the background hydrolysis obtained with the same amount of GST, used as a negative control ( Figure 1). Similar results were obtained with an alternative substrate, resorufin β-Dglucopyranoside [26]. There are two glutamic acid residues in SrfJ, Glu196 and Glu294, which, according to the crystal structure of the protein [24], are located at positions equivalent to the catalytic residues Glu235 and Glu340 of human glucosylceramidase. To test the hypothesis that these residues are also relevant for the activity of SrfJ, three different mutants were obtained in the 6His fusion plasmid to generate the proteins 6His-SrfJE196A, 6His-SrfJE294A, and 6His-SrfJE196AE294A ( Figure S1), in which one or both putative catalytic glutamic acid residues were changed into alanines. All these mutants completely lost activity since they produced the same level of fluorescence in the in vitro assay as the protein used as a negative control ( Figure 1). These results, together with similarity data, suggest that SrfJ possesses glucosylceramidase activity and that Glu196 and Glu294 are essential for this function.

Glucosylceramidase Activity of SrfJ
The similarity between SrfJ and human glucosylceramidase led us to explore the enzymatic activity of this Salmonella effector. For this purpose, the 4-methylumbelliferylβ-D-glucopyranoside substrate was used. Hydrolysis of this substrate results in a product with emission at the blue region of the fluorescence spectrum. The purified 6His-SrfJ in this assay showed a glucosidase activity of 0.83 U/mg, significantly higher than the background hydrolysis obtained with the same amount of GST, used as a negative control ( Figure 1). Similar results were obtained with an alternative substrate, resorufin β-Dglucopyranoside [26]. There are two glutamic acid residues in SrfJ, Glu196 and Glu294, which, according to the crystal structure of the protein [24], are located at positions equivalent to the catalytic residues Glu235 and Glu340 of human glucosylceramidase. To test the hypothesis that these residues are also relevant for the activity of SrfJ, three different mutants were obtained in the 6His fusion plasmid to generate the proteins 6His-SrfJE196A, 6His-SrfJE294A, and 6His-SrfJE196AE294A ( Figure S1), in which one or both putative catalytic glutamic acid residues were changed into alanines. All these mutants completely lost activity since they produced the same level of fluorescence in the in vitro assay as the protein used as a negative control ( Figure 1). These results, together with similarity data, suggest that SrfJ possesses glucosylceramidase activity and that Glu196 and Glu294 are essential for this function. SrfJ is a glucosylceramidase. The activity of the indicated proteins was measured by their ability to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside. Specific activity was calculated as pmol of product produced per min and µg of protein using 4-methylumbelliferone as a calibration standard. Values are means + standard deviations. n = 3.

Effect of SrfJ on the Host Lipidome
The in vitro activity shown for SrfJ in the previous section suggested that this Salmonella effector could have a role in the manipulation of lipids in the host cell. To test this hypothesis, we transfected human HEK293T cells either with a plasmid producing SrfJ or with the empty vector, and lipids were extracted and analyzed using highthroughput lipidome profiling mass spectrometry. This analysis detected 917 compounds in positive ionization mode and 933 in negative ionization mode. Multivariate statistics (principal component analysis) showed a clear alteration of the lipidome induced by the presence of SrfJ, with clear separation between control and SrfJ-expressing cells for lipids with negative ionization (Figure 2). SrfJ is a glucosylceramidase. The activity of the indicated proteins was measured by their ability to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside. Specific activity was calculated as pmol of product produced per min and µg of protein using 4-methylumbelliferone as a calibration standard. Values are means + standard deviations. n = 3.

Effect of SrfJ on the Host Lipidome
The in vitro activity shown for SrfJ in the previous section suggested that this Salmonella effector could have a role in the manipulation of lipids in the host cell. To test this hypothesis, we transfected human HEK293T cells either with a plasmid producing SrfJ or with the empty vector, and lipids were extracted and analyzed using high-throughput lipidome profiling mass spectrometry. This analysis detected 917 compounds in positive ionization mode and 933 in negative ionization mode. Multivariate statistics (principal component analysis) showed a clear alteration of the lipidome induced by the presence of SrfJ, with clear separation between control and SrfJ-expressing cells for lipids with negative ionization (Figure 2). Univariate statistics (t-test, p < 0.05) revealed 64 lipid species (42 in positive ionization mode and 22 in negative ionization mode) with significantly different levels between both conditions (Table S1). We were able to determine the identity of some of them based on exact mass, retention time, isotopic distribution, and MS/MS spectrum. This analysis identified seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ (Table 1 and Figure 3).  Univariate statistics (t-test, p < 0.05) revealed 64 lipid species (42 in positive ionization mode and 22 in negative ionization mode) with significantly different levels between both conditions (Table S1). We were able to determine the identity of some of them based on exact mass, retention time, isotopic distribution, and MS/MS spectrum. This analysis identified seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ (Table 1 and Figure 3).     Interestingly, consistent with the glucosylceramidase activity of SrfJ, one of the lipids that was over-represented in the presence of SrfJ was a ceramide. Then, we decided to perform a targeted analysis of ceramides and hexosylceramides (which include glucosylceramides and galactosylceramides, that cannot be distinguished with these methods) comparing HEK293T cells transfected with pcDNA3 (empty vector) or pIZ1855 (pcDNA3-SrfJ-3xFLAG). This analysis revealed an impact of SrfJ on the levels of these sphingolipids ( Figure 4A,B). Importantly, the Cer/HexCer ratio was increased in SrfJ-expressing cells, and this increase was statistically significant for the specific species 34:1 and 36:1, as well as for the total amount of these lipids ( Figure 4C). Interestingly, consistent with the glucosylceramidase activity of SrfJ, one of the lipids that was over-represented in the presence of SrfJ was a ceramide. Then, we decided to perform a targeted analysis of ceramides and hexosylceramides (which include glucosylceramides and galactosylceramides, that cannot be distinguished with these methods) comparing HEK293T cells transfected with pcDNA3 (empty vector) or pIZ1855 (pcDNA3-SrfJ-3xFLAG). This analysis revealed an impact of SrfJ on the levels of these sphingolipids ( Figure 4A,B). Importantly, the Cer/HexCer ratio was increased in SrfJexpressing cells, and this increase was statistically significant for the specific species 34:1 and 36:1, as well as for the total amount of these lipids ( Figure 4C). To obtain additional data on the effect of SrfJ on the host lipidome under physiological conditions, we infected RAW264.7 murine macrophages with either wildtype Salmonella or a ΔsrfJ strain and analyzed the abundance of ceramides in these cells at 8 h post-infection and in uninfected cells. As seen in Figure 5, infection with Salmonella causes a decrease in glycosylated ceramides and an increase in the relative abundance of To obtain additional data on the effect of SrfJ on the host lipidome under physiological conditions, we infected RAW264.7 murine macrophages with either wild-type Salmonella or a ∆srfJ strain and analyzed the abundance of ceramides in these cells at 8 h postinfection and in uninfected cells. As seen in Figure 5, infection with Salmonella causes a decrease in glycosylated ceramides and an increase in the relative abundance of ceramides.
Interestingly, the ratio Cer/HexCer is higher in wild-type infected cells than in cells infected with a srfJ mutant, suggesting that SrfJ plays a role in the generation of the altered patterns observed in host lipids.
ceramides. Interestingly, the ratio Cer/HexCer is higher in wild-type infected cells than in cells infected with a srfJ mutant, suggesting that SrfJ plays a role in the generation of the altered patterns observed in host lipids. Figure 5. Effect of Salmonella infection on the ratios of ceramide/hexosylceramide in RAW264.7 macrophages. Total ceramide/hexosylceramide ratios in RAW264.7 macrophages not infected, infected with wild-type S. enterica serovar Typhimurium strain 14028, or infected with a ΔsrfJ mutant. Data are presented as mean values + standard deviations of twelve biological replicates. Differences were considered significant if p < 0.05 (Student's t test). * p < 0.05, ** p < 0.01, ns: not significant, for the indicated comparisons.

Effect of SrfJ on the Host Transcriptome
Ceramides are involved in several important signal transduction pathways [27,28]. Therefore, we reasoned that alterations in the proportions of sphingolipids induced by SrfJ may also have an impact on the expression of host genes. To study this possibility, we performed a transcriptomic analysis using two different approaches. First, we analyzed the specific effect of the ectopic expression of SrfJ on epithelial human HeLa cells. RNA was obtained from HeLa cultures transiently transfected with either pcDNA3-SrfJ-3xFLAG or pcDNA3 (empty vector), and the analysis of their transcriptomes was performed using Clariom S Assay, HUMAN microarrays (Affymetrix, Santa Clara, CA, USA). Figure 6A represents the 1201 genes with statistically significant (p < 0.05) differential expression in SrfJ-transfected compared to HeLa cells transfected with the empty vector. The 45 differentially expressed genes with a linear fold change threshold of 2 or more for upregulated genes, 0.5 or less for downregulated genes, are described in Table S2. For a more physiological approach, we compared the transcriptomes of RAW264.7 murine macrophages infected for 8 h by either wild-type Salmonella or a srfJ mutant derivative, using Clariom S Assay, MOUSE microarrays (Affymetrix, Santa Clara, CA, USA) to compare transcriptomes. Genes with statistically significant (p < 0.05) differential expression in this comparison are represented in Figure 6B. Among 1408 differentially expressed genes, 68 overcame a linear fold change threshold of 2 for upregulated genes, 0.5 for downregulated genes (excluding predicted genes of unknown function). These genes are described in Table S3. Data are presented as mean values + standard deviations of twelve biological replicates. Differences were considered significant if p < 0.05 (Student's t test). * p < 0.05, ** p < 0.01, ns: not significant, for the indicated comparisons.

Effect of SrfJ on the Host Transcriptome
Ceramides are involved in several important signal transduction pathways [27,28]. Therefore, we reasoned that alterations in the proportions of sphingolipids induced by SrfJ may also have an impact on the expression of host genes. To study this possibility, we performed a transcriptomic analysis using two different approaches. First, we analyzed the specific effect of the ectopic expression of SrfJ on epithelial human HeLa cells. RNA was obtained from HeLa cultures transiently transfected with either pcDNA3-SrfJ-3xFLAG or pcDNA3 (empty vector), and the analysis of their transcriptomes was performed using Clariom S Assay, HUMAN microarrays (Affymetrix, Santa Clara, CA, USA). Figure 6A represents the 1201 genes with statistically significant (p < 0.05) differential expression in SrfJ-transfected compared to HeLa cells transfected with the empty vector. The 45 differentially expressed genes with a linear fold change threshold of 2 or more for upregulated genes, 0.5 or less for downregulated genes, are described in Table S2. For a more physiological approach, we compared the transcriptomes of RAW264.7 murine macrophages infected for 8 h by either wild-type Salmonella or a srfJ mutant derivative, using Clariom S Assay, MOUSE microarrays (Affymetrix, Santa Clara, CA, USA) to compare transcriptomes. Genes with statistically significant (p < 0.05) differential expression in this comparison are represented in Figure 6B. Among 1408 differentially expressed genes, 68 overcame a linear fold change threshold of 2 for upregulated genes, 0.5 for downregulated genes (excluding predicted genes of unknown function). These genes are described in Table S3.

Functions of Genes Differentially Expressed in the Presence of SrfJ
To gain insight into cell processes affected by SrfJ, we explored the sets of differentially expressed genes using ShinyGO version 0.77 [29]. This analysis detected a significant enrichment of gene ontology (GO) terms in the database of biological processes for the set of genes that were differentially expressed in HeLa cells producing SrfJ ( Figure 7A). Among the processes involved are the inflammatory response and the response to different stimuli, including molecules of bacterial origin. The network of interactions between proteins encoded by these genes was obtained using STRING [30] and is depicted in Figure 7B.

Functions of Genes Differentially Expressed in the Presence of SrfJ
To gain insight into cell processes affected by SrfJ, we explored the sets of differentially expressed genes using ShinyGO version 0.77 [29]. This analysis detected a significant enrichment of gene ontology (GO) terms in the database of biological processes for the set of genes that were differentially expressed in HeLa cells producing SrfJ ( Figure  7A). Among the processes involved are the inflammatory response and the response to different stimuli, including molecules of bacterial origin. The network of interactions between proteins encoded by these genes was obtained using STRING [30] and is depicted in Figure 7B.  A similar analysis showed that some biological processes were overrepresented in the set of differentially expressed genes in the comparison between RAW264.7 cells infected with wild-type Salmonella and RAW264.7 cells infected with the srfJ mutant ( Figure 8). A similar analysis showed that some biological processes were overrepresented in the set of differentially expressed genes in the comparison between RAW264.7 cells infected with wild-type Salmonella and RAW264.7 cells infected with the srfJ mutant ( Figure 8).  Interestingly, some common processes were detected to be enriched in both sets of differentially expressed genes, including the inflammatory response and the response to different stimuli.

Secretion of CCL5 by Host Cells Is Modulated by SrfJ
The only gene the expression of which changed more than two-fold in the presence of SrfJ both in transfected HeLa cells and in infected RAW264.7 cells was CCL5. The product of this gene, CCL5, also known as RANTES, is a chemokine that induces leukocyte migration and can mediate inflammatory responses [31]. To explore the possibility that SrfJ may modulate the secretion of CCL5 in host cells, we infected RAW264.7 macrophages with wild-type S. enterica serovar Typhimurium or with a ∆srfJ mutant and measured the concentration of the chemokine in the supernatants of cell cultures 8 h post-infection. As seen in Figure 9A, Salmonella infection triggers the secretion of CCL5 by macrophages as expected, but the level of secretion increases when infecting with an ∆srfJ mutant, suggesting that SrfJ contributes to the modulate secretion of this chemokine. To confirm the effect of SrfJ on CCL5 secretion, human HeLa cells and HEK293T cells were transfected with a plasmid expressing SrfJ or with the empty vector as a control. The results shown in Figure 9B,C indicate that SrfJ very significantly decreases the secretion of CCL5.

Discussion
Most T3SS effectors target host proteins and there have been many studies describing the roles of these interactions in Salmonella virulence. On the contrary, studies relating effectors to host lipids are scarce. However, the subversion of host lipid signaling is essential for successful infection [32]. Phosphoinositides are particularly important during phagosome maturation, and Salmonella manipulates their metabolism through the T3SS effector SopB. This effector functions as a phosphoinositide phosphatase and phosphotransferase [33]. Indeed, there have been reports showing that S. enterica serovar Typhimurium infection results in an increase in intracellular cholesterol content in a T3SS2-dependent manner. For instance, the effectors SseJ and SseL have roles in cholesterol metabolism and transport. SseJ is a RhoA-dependent cholesterol acyltransferase, the activity of which is necessary to suppress the expression of the gene encoding the ABCA1 transporter through a signaling cascade that involves FAK and Akt kinases [34]. Both SseJ and SseL interact with the host cholesterol transport protein OSBP1 [35,36].
SrfJ shares 30% amino acid sequence identity with human glucosylceramidase on its 447 residues, and their structures are highly similar [24]. These similarities suggested that SrfJ could also be a glucosylceramidase. Here, using an in vitro assay, we have shown that SrfJ indeed possesses this activity, also showing the essential role of the predicted catalytic residues Glu196 and Glu294 (Figure 1). To the best of our knowledge, this is the first T3SS effector shown to have glucosylceramidase activity. The expression of srfJ causes significant changes in the lipidome of human cells, which are compatible with the activity observed in vitro, since the ratios HexCer/Cer are increased (Figure 4), suggesting that SrfJ is also active as a glucosylceramidase in vivo. Importantly, these results are confirmed in the context of host cell infections. Salmonella infection increases the HexCer/Cer ratio of murine macrophages in a SrfJ-dependent manner ( Figure 5). The effect of SrfJ in this context is not related to differential intracellular survival, since our previous results showed that the srfJ mutant has no significant defect in intracellular proliferation compared to the wild-type strain [20]. In addition to Cer and HexCer, the abundance of many other lipids is altered by the presence of SrfJ, which could be due either to indirect effects or to other unknown activities of this effector.
Ceramide is the precursor of all complex sphingolipids. These are important signaling molecules that regulate numerous physiological and pathological processes, including signal transduction, the regulation of cell growth and death, adhesion, migration, immunity, and inflammation [37,38]. Bioactive sphingolipids are involved in the regulation of the internalization of bacteria in the host cell and the induction of an immunological response. However, some bacterial pathogens are capable of interfering with sphingolipid signaling by hijacking specific host cell factors or producing enzymes that are involved in sphingolipid metabolism [39]. Examples of the latter are sphingomyelinases of Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, Mycobacterium tuberculosis and Legionella pneumophila, ceramidase and sphingomyelin synthase of Pseudomonas aeruginosa, sphingosine phosphate lyase of Burkholderia pseudomallei, Burkholderia thailandensis and L. pneumophila, and sphingosine kinase from L. pneumophila.
Since ceramide and other derived sphingolipids are involved in the control of several signaling pathways that may ultimately affect the transcription of host genes, we decided to explore the effects of SrfJ on the host transcriptome. This analysis revealed that the levels of transcriptional products of many genes are significantly altered by SrfJ both when expressed alone in epithelial cells and in the context of macrophage infections ( Figure 6, Tables S2 and S3). Interestingly, the enrichment analysis points to genes involved in the innate immune response and inflammation among those modulated by SrfJ ( Figures 7A and 8A). Importantly, this analysis was validated by our experiments, showing a small but significant increase in the secretion of the chemokine CCL5 from macrophages that were infected with a srfJ mutant strain of Salmonella compared to those that were infected with the wild-type strain ( Figure 9A). These results were confirmed by transfection experiments in which the presence of SrfJ very significantly reduced the secretion of CCL5 in human cells ( Figure 9B,C). This cytokine functions as a chemoattractant for blood monocytes, memory T helper cells, and eosinophils, causing the release of histamine from basophils, and activating eosinophils [40]. Our results suggest that SrfJ may contribute to a moderation of the level of host cytokine secretion induced by Salmonella infection, and thus protect bacteria from host immune defenses.
In summary, this work reveals for the first time the existence of a T3SS effector with glucosylceramidase activity, the presence of which in host cells results in significant alterations of lipid patterns and gene expression that may have immunomodulatory consequences in the host.

DNA Amplification with Polymerase Chain Reaction and Sequencing
Amplification reactions were carried out on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) using Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) or MyTaq Red DNA polymerase (Bioline Memphis, TN, USA) according to the supplier's instructions. Oligonucleotides are described in Table 3. The constructs were sequenced with an automated DNA sequencer (Stab Vida, Oeiras, Portugal).

Bacterial Culture
The standard culture medium for S. enterica and Escherichia coli was LB broth. Solid LB contained agar 1.5% final concentration. Antibiotics were used at the following concentrations: kanamycin (Km), 50 µg/mL; ampicillin (Ap), 100 µg/mL.

Mutagenesis
The disruption of srfJ and the replacement with a gene conferring Km resistance was carried out as previously described [45] using primers srfJP1 and srfJP2 and the plasmid pKD13. The antibiotic resistance cassette introduced by the gene targeting procedure was eliminated by recombination using the FLP helper plasmid pCP20.
To generate a point mutation that affects the putative catalytic residue Glu196 of SrfJ, the plasmid pIZ2046 (pQE30-SrfJ) was used as a template for PCR amplification using the primer pair srfJE196Afw/srfJE196Arv. The products were digested with 1 µL of DpnI (10 U/µL) for 1 h at 37 • C and used to transform E. coli XL1-Blue. Overlap extension PCR was used to obtain a similar mutation that affects residue Glu294 using primer pairs srfJBamfw/srfJE294Arv, srfJE294Afw/srfJSalrv, and srfJBamfw/srfJSalrv. The final product was digested with BamHI and SalI and ligated to pQE30 digested with the same endonucleases. Mutations were verified by Sanger sequencing.

GST and 6His Fusion Proteins
GST expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to bacteria containing pGEX-4T-1, and the protein was isolated from bacterial lysates by affinity chromatography with glutathione-agarose beads (Sigma-Aldrich, San Luis, MO, USA). For lysis, the bacteria were sonicated in NP40 buffer. Then, 6His fusion proteins were produced after the addition of 1 mM isopropyl-β-D-thiogalactoside to E. coli XL1-Blue containing derivatives of pQE30, purified in Ni-NTA agarose beads (Sigma-Aldrich), and eluted with 300 mM imidazole in binding buffer (50 mM NaH 2 PO 4 , 300 mM NaCl).

Glucosylceramidase Assay
The activity of the recombinant proteins was measured by their ability to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside [46,47]. The SrfJ proteins (wild-type and mutants) were purified as 6His fusion proteins and diluted in assay buffer (50 mM sodium citrate, 25 mM sodium cholate, 5 mM DTT, pH 6.0). The reaction was started by mixing 25 µL of the diluted proteins with 25 µL of 6 mM substrate (4-methylumbelliferyl-β-Dglucopyranoside) prepared in assay buffer. After 20 min of incubation at 37 • C, the reaction was stopped by adding 50 µL of stop solution (0.5 M glycine, 0.3 M NaOH, pH 10.0). Fluorescence was measured at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode. Specific activity was calculated as pmol of product produced per min and µg of protein using 4-methylumbelliferone as a calibration standard.

Infections of RAW264.7 Cells with Salmonella
RAW264.7 cells were seeded in 24-well plates at 1.5 × 10 5 cells per well and incubated for 24 h at 37 • C with 5% CO 2 . The bacteria were grown in LB medium for 24 h at 37 • C with shaking and added at a multiplicity of infection of 250. Cell culture was washed twice with phosphate buffered saline (PBS) 1 h after infection, overlaid with DMEM containing 100 µg/mL gentamicin, and incubated for another hour. The culture was then washed twice with PBS, covered with DMEM with gentamicin (16 µg/mL), and incubated for 6 h. Infected RAW264.7 cells showed a 65% viability compared to non-infected cells. This viability was similar using the srfJ strain and wild-type Salmonella.

Lipidomic Analysis
Untargeted lipidomic analysis was carried out at the Lipidomics Platform of the Biomedical Research Institute of Lleida (Lleida, Spain). Lipids were extracted from transfected HEK293T cells (one million cells per sample, six samples per condition) using an MTBE-based method, as previously described [48]. Lipid extracts were subjected to liquid chromatography coupled to mass spectrometry (LC-MS) using an Agilent UPLC 1290 coupled to the Q-TOF MS/MS 6520 (Agilent Technologies, Santa Clara, CA, USA) based on previously published methods [49,50]. Duplicate runs of each sample were performed to collect positive and negative electrospray ionized lipid species. The identity of relevant lipid species was confirmed by exact mass, retention time, and/or MS/MS spectrum, as previously described [51].
The targeted lipidomic analysis was performed by the Biomolecular Mass Spectrometry (BIO-MS) service of the University Pablo de Olavide (Seville, Spain). For this analysis, each cell pellet was resuspended in 1 mL of MeOH/MTBE/CHCl 3 (1.33:1:1, v/v/v, MTBE stands for methyl tert-butyl ether) with added internal standards (Cer(35:1), HexCer(35:1), Hex2Cer(35:1)), and vortexed for 20 s. Subsequently, the mixture was incubated in a shaker at 900 rpm for 1 h at room temperature (RT). The sample was then sonicated for 30 min and incubated for 20 min at RT. Particulate matter was pelleted by centrifugation at 16,000× g for 10 min at 20 • C. The supernatant was collected and dried in a vacuum centrifuge for 120 min at 30 • C. The extracted lipids were reconstituted in 60 µL butanol:water (1:1, v/v) and sonicated for 10 min. Then, 60 µL of methanol with 10 mM ammonium formate was added and the samples were centrifuged (1711 g, 6 min, 20 • C). The supernatants were then transferred to a 0.2 mL glass vial for UHPLC-MS/MS analysis. Lipidomic analysis was performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) using a Thermo Scientific-Dionex Ultimate 3000 UHPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer. The lipids were separated by reversed-phase chromatography using a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA USA). Solvents A and B consisted of tetrahydrofuran:methanol:water in ratios 20:20:60 and 75:20:5, respectively, both containing 10 mM ammonium formate. The column and autosampler temperatures were regulated at 50 and 25 • C, respectively. The following gradient conditions were used: (flow rate 400 µL/min) 0% solvent B to 40% solvent B for 2 min, 40% solvent B to 100% solvent B for 6.5 min, 0.5 min at 100% solvent B, and a return to 0% solvent B for 0.5 min, and then 0.5 min at 0% solvent B prior to the next injection. The liquid chromatography eluent was then analyzed by ESI-MS/MS using full MS/ddMs 2 scan mode. Mass spectrometry detection was performed using a Q Exactive™. Lipids were detected by electrospray ionization in positive mode with a mass resolution of 70,000. Nitrogen was used as a desolvation gas. Data were acquired in the m/z range from 150 to 1500. Electrospray ionization parameters were as follows: sheat gas flow 45 arb (arbitrary units), auxiliary gas flow 8 arb, auxiliary gas heater temperature 350 • C, spray voltage 3.5 kV, capillary temperature 320 • C.
For statistical analyses of lipidomic analyses, Mass-Hunter Mass Profiler Professional software (Agilent Technologies, Santa Clara, CA, USA) and Metaboanalyst software version 5.0 [52] were used. Other statistical calculations were performed using SPSS software version 26 (Chicago, IL, USA). Differences in the number of molecules between conditions were analyzed using a t-test. A level of p < 0.05 was considered statistically significant.

RNA Preparation, Gene Array Processing, and Statistics
Total RNA from HeLa or RAW264.7 cells was isolated using 1 mL of TRIzol reagent (Invitrogen) according to the manufacturer's instructions. An additional purification step was performed with the RNeasy Min Elute Cleanup Kit (Qiagen, Hilden, Germany). Biotinylated single-stranded cDNA was prepared from 100 ng per sample of total intact RNA (3 samples per condition). Labeled cDNA was hybridized with the Clariom S Assay, HUMAN arrays (for HeLa RNA) or Clariom S Assay, MOUSE arrays (for RAW264.7 RNA) (Affymetrix) following the manufacturer's instructions. The arrays were scanned on a 3000 7G scanner (Affymetrix). Image analysis, fluorescent data quantification, and quality control were performed with Affymetrix software, the GeneChip Command Console version 2.0, and the Transcriptome Analysis Console version 4.0.2. All procedures and preliminary data analysis were performed in the Genomics Unit of the Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER, Seville, Spain). Statistical significance (p-value) was calculated using the empirical Bayes moderated t test based on the results of three arrays per condition. The microarray data used in this analysis are available from the NCBI Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo/ (accessed on 7 October 2022) under accession numbers GSE215051 and GSE215053.

Detection of CCL5 Secretion by ELISA
Supernatants of murine RAW264.7 cells either non-infected or infected with wild-type or a srfJ mutant Salmonella, as well as supernatants of transfected human HeLa or HEK293T cells, were assayed for CCL5 secretion using the mouse or the human CCL5/RANTES DuoSet ELISA kits (R&D SYSTEMS) following manufacturer's instructions. Briefly, 96-well polysorbent plates previously incubated overnight at room temperature with mouse CCL5 capture antibody were washed, and then blocked with Reagent Diluent for 2 h at room temperature and washed again. Then, 100 µL of appropriately diluted samples of supernatant (and serial dilutions of an appropriate mouse CCL5 standard, for CCL5 quantification) were transferred to the plates and incubated for 2 h at room temperature. Then, plates were washed again, followed by 2 h of incubation at room temperature with CCL5 detection antibody, a new wash, and 20 min of incubation, at room temperature and in the dark, with streptavidin-HRP B. Plates were then washed one final time, and incubated for 20 min at room temperature in the dark with substrate solution (TMB ELISA Substrate, R&D), upon which STOP solution was added, and signal development and detection were carried out using a Synergy HT plate reader (Biotek, Winooski, VT, USA) at a wavelength of 450 nm, with a subtraction wavelength of 570 nm. Serially diluted CCL5 standards were used for the extrapolation of protein concentration from absorbance data. Funding: This research was funded by grant PID2019-106132RB-I00 funded by MCIN/AEI/10.13039/ 501100011033. It was also funded by Fondo Europeo de Desarrollo Regional (FEDER) y Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía: grant P20_00576; Universidad de Sevilla, Fondo Europeo de Desarrollo Regional (FEDER) y Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía: grant US-1380805; Plan de Recuperación, Transformación y Resiliencia «European Union-NextGenerationEU» (via a María Zambrano research fellowship, to RB).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are openly available from NCBI Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo/ (accessed on 7 October 2022) under accession numbers GSE215051 and GSE215053.